TY - JOUR
T1 - Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering
AU - Zhao, Evan M.
AU - Lalwani, Makoto A.
AU - Lovelett, Robert J.
AU - Garciá-Echauri, Sergio A.
AU - Hoffman, Shannon M.
AU - Gonzalez, Christopher L.
AU - Toettcher, Jared E.
AU - Kevrekidis, Ioannis G.
AU - Avalos, José L.
N1 - Funding Information:
We thank all members of the Avalos, Kevrekidis, and Toettcher laboratories for helpful comments. We thank Dr. Christina DeCoste, Dr. Katherine Rittenbach, and the Princeton Molecular Biology Flow Cytometry Resource Center for assistance with flow cytometry experiments. We thank Dr. Kris Prather for pRS304 and kIura3mb-7xtetOpr, as well as Dr. Joerg Stelling for FRP467-PACT1(-1-520)-LexA-ER-haVP16, FRP880_PACT1(-1-520)-LexA-ER-haB112-TCYC1, and FRP795_insul-(lexA-box)8-PminCYC1-Citrine-TCYC1. This work was supported by the Maeder Graduate Fellowship in Energy and the Environment (to EMZ), The Pew Charitable Trusts, the U.S. DOE Office of Biological and Environmental Research, Genomic Science Program Award DE-SC0019363, and NSF CAREER Award CBET-1751840 (to JLA), the NIH grant DP2EB024247 (to JET) and a Schmidt Transformative Technology grant (to JET, IGK and JLA), and the DARPA Lagrange Program, Contract no. N66001-18-C-4031 (RJL and IGK).
Publisher Copyright:
©
PY - 2020/12/18
Y1 - 2020/12/18
N2 - The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in Saccharomyces cerevisiae. The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible GAL1 promoter (PGAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with PGAL1-S achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and PGAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.
AB - The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in Saccharomyces cerevisiae. The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible GAL1 promoter (PGAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with PGAL1-S achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and PGAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.
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U2 - 10.1021/acssynbio.0c00305
DO - 10.1021/acssynbio.0c00305
M3 - Article
C2 - 33232598
AN - SCOPUS:85097881486
SN - 2161-5063
VL - 9
SP - 3254
EP - 3266
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 12
ER -